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Methods, devices, arrays and kits for detecting and analyzing biomolecules

a biomolecule and array technology, applied in the field of biomolecule detection and analysis methods, can solve the problems of low biomolecule specificity and high affinity of certain membranes used in such methods, and achieve the effect of increasing specificity

Inactive Publication Date: 2004-04-29
HEALTH & HUMAN SERVICES THE US SEC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0026] Also provided are kits for replicating a pattern of biomolecules from a tissue sample, which kits include a plurality of membranes, each having a coating on its upper and / or lower surfaces to increase specific binding of a target biomolecule, a quantity of transfer buffer, and a fluid impervious enclosure (for instance, a heat-sealable bag).
0027] Another example of a described kit is a kit for analyzing a proteome, which kit contains a plurality of membranes, each having a affinity for at least one protein, and a plurality of reagent species (such as detector molecules, particularly labeled detectors), each species is adapted to detect one or more specific proteins bound to the membranes.

Problems solved by technology

Certain membranes for use in such methods have a high affinity but low capacity for biomolecules, for instances proteins, nucleic acids, lipids, carbohydrates, or combinations thereof.

Method used

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  • Methods, devices, arrays and kits for detecting and analyzing biomolecules
  • Methods, devices, arrays and kits for detecting and analyzing biomolecules
  • Methods, devices, arrays and kits for detecting and analyzing biomolecules

Examples

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first embodiment

[0155] More specifically, in a first embodiment, membrane stack 13 comprises one or more membranes 12, for instance up to five membranes, generally constructed as described herein. The membranes 12 in stack 13 should have a high affinity for proteins and other biomolecules but have a low capacity for retaining such molecules. This feature permits the molecules to pass through the membrane stack with only a limited number being trapped on each of the successive layers, thereby allowing multiple "carbon copies" (substantial copies that are not necessarily identical copies, they may have slight differences but can be identical or nearly identical) to be generated. In other words, the low capacity allows the creation of multiple replicates as only a limited quantity of the biomolecules are trapped on each layer.

[0156] First and second filter pads 24, 26 are preferably constructed of a blotting paper such as GB004 Blotter Paper available from Schleicher and Schuell. Filter pads 24, 26 ar...

second embodiment

[0230] Thus, with another embodiment, each of the membranes will bind to a different group of proteins essentially permitting "3-D gel electrophoresis" by allowing proteins to be separated into three dimensions: in the X and Y dimensions by charge and mass, and then in the Z dimension by an additional chemical characteristic. The proteins on the membranes can be visualized by the immuno-staining and imaging methods set forth below. They may also be advantageously analyzed by mass spectrometry either without additional cleavage or after such cleavage (see, e.g., WO00 045168), or by other means. Examples of the methods and kits facilitate such analysis because the stratification by the different membranes helps to expose moderate and low abundance protein spots that would otherwise be undetectable on standard 2-D gels. The more spots that are available for analysis, the more data can be generated by mass spectroscopy or by such other approaches.

[0231] Other Membrane Characteristics

[0...

example detection

[0257] Example Detection Chemistries with Detector Cocktails

[0258] In certain embodiments, after proteins have been transferred through the membrane stack, individual membranes layers are separated and each is incubated in a separate antibody (or other detector molecule) cocktail. A key advantage of creating multiple replicate blots is that many more detector molecules (e.g., antibodies) can be usefully employed than if all of the detectors had to be crowded onto a single blot.

[0259] An exemplary process for designing the ligand cocktails--and for determining which proteins will be identified on each membrane layer--is provided below. First the panel of proteins of interest is selected. These can be randomly selected proteins and / or proteins that are not directly related to one another or may be groups of known proteins previously implicated to play a role in one or more particular cellular phenomena (e.g. apoptosis, cell cycle progression) or a particular disease (e.g. prostate can...

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Abstract

The present disclosure is directed to devices, arrays, kits and methods for detecting biomolecules in a tissue section (such as a fresh or archival sample, tissue microarray, or cells harvested by an LCM procedure) or other substantially two-dimensional sample (such as an electrophoretic gel or cDNA microarray) by creating "carbon copies" of the biomolecules eluted from the sample and visualizing the biomolecules on the copies using one or more detector molecules (e.g., antibodies or DNA probes) having specific affinity for the biomolecules of interest. Specific methods are provided for identifying the pattern of biomolecules (e.g., proteins and nucleic acids) in the samples. Other specific methods are provided for the identification and analysis of proteins and other biological molecules produced by cells and / or tissue, especially human cells and / or tissue. The disclosure also provides a plurality of differentially prepared and / or processed membranes that can be used in described methods, and which permit the identification and analysis of biomolecules.

Description

REFERENCE TO RELATED APPLICATIONS[0001] This application is a Continuation in Part (CIP) of U.S. patent application Ser. No. 09 / 753,574 (filed Jan. 4, 2001), which is a CIP of U.S. patent application Ser. No. 09 / 718,990 (Filed Nov. 20, 2000), which is a CIP of International Patent Application No. US00 / 20354, filed Jul. 26, 2000 and published in the English language, and claims the benefit of U.S. Provisional Patent Application No. 60 / 145,613 (filed Jul. 26, 1999). The current application further claims the benefit of U.S. Provisional Patent Application Nos. 60 / 286,258 (filed Apr. 25, 2001), 60 / 304,031 (filed Jul. 9, 2001), and 60 / 296,475 (filed Jun. 8, 2001). Each of these related applications is incorporated herein in their entirety.FIELD OF THE DISCLOSURE[0003] The present disclosure is directed to methods, devices, arrays, and kits for identifying and analyzing large numbers of biomolecules in a sample, such as a biological sample. The disclosure further relates to using these me...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): B01L3/00G01N1/28G01N33/543
CPCB01L3/5023G01N1/2813G01N27/44739G01N2001/284G01N2001/368G01N33/54386
Inventor KNEZEVIC, VLADIMIREMMERT-BUCK, MICHAEL RBAIBAKOVA, GALINAHARTMANN, DAN-PAULHEWITT, STEPHEN MMITCHELL, CAPRE DENISEGARDNER, KEVIN
Owner HEALTH & HUMAN SERVICES THE US SEC
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